Team:Heidelberg/Notebook/Killing I/Notebook/week11

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Latest revision as of 14:38, 29 October 2008

Week 11

Monday, 10/13/08

Proceeding of phage cloning strategy two

• Digestion of the miniprep 1-6,9,10 from sunday and the original pBluescript with insert

• Digestion with XbaI/XhoI (top)
  • normal: 4549, 2157, 34
  • new: 3945, 2898

• Digestion with SacI/SpeI (bottom)
  • normal: 4549, 2157, 34
  • new: 4549, 1331, 929, 34

• Gel

• • lane0: dna ladder mix
  • lane1: sample 1
  • lane2: sample 2
  • lane3: sample 3
  • lane4: sample 4
  • lane5: sample 5
  • lane6: sample 6
  • lane9: sample 9
  • lane10: sample 10
  • lane11: pBluescript with insert

• PCR with CmR_suffix and CmR_prefix (top)
  • normal: 1668bp
  • new: 852bp, 919bp

• PCR with oriT_prefix and CmR_suffix (bottom)
  • normal: 2150bp
  • new: 1329bp

• Gel

• • lane0: dna ladder mix
  • lane1: sample 1
  • lane2: sample 2
  • lane3: sample 3
  • lane4: sample 4
  • lane5: sample 5
  • lane6: sample 6
  • lane9: sample 9
  • lane10: sample 10
  • lane11: pBluescript with insert

• --> we do not have a working GFP/CmR in pBlue/insert!!! --> do the ligation again (beginng from the mutagenesis pcr)

Proceeding of cloning CmR and oriT in standard plasmid

• inoculation of CmR Std. Mutagenesis PCR sample and oriT Std. colonies

Tuesday, 10/14/08

Proceeding of cloning CmR and oriT in standard plasmid

• Miniprep of 6 oriT and 5 CmR Std. samples
  • digestion with EcoRI/PstI (to cut out insert of pSB1A2)

• • lane0: dna ladder mix
  • lane1-6: oriT 1-6
  • lane7: 1kb dna ladder plus
  • lane8: CmR Std. Mut 1.1.1
  • lane9: CmR Std. Mut 1.1.2
  • lane10: CmR Std. Mut 1.2.2
  • lane11: CmR Std. Mut 2.2.1
  • lane12: CmR Std. Mut 2.2.2

• • expected fragments:
    • oriT: 2000bp, 500bp
    • CmR: 2000bp, ca. 900bp

• -->no oriT is right

• -->CmR 1.1.1, 1.1.2, 2.2.1, 2.2.2 look good
• -->sequencing of 2.2.1 and 2.2.2
  • sequencing results match perfect

• Three ligations of oriT pcr product with pSB1A2 backbone (PstI, XbaI) (1:2.5, 2:5, 3:7.5 (µl, vector:insert))
  • 40min at RT
  • transformation, plated out on Amp plates

• Screening PCR of oriT agar plates
  • pcr with standard plasmid primers (VF2, VR) using Taq
  • 12 pcr samples (1-6 only one colony, 7-12 three colonies)
  • Gel

• • lane0: dna ladder mix
  • lane1-12: screening sample 1-12
  • expected fragment length: ca. 500-600bp
• -->sample 3 and 5 look good
• -->inoculation of overnight cultures
• -->sequencing of 3 and 5
  • sequencing results match perfect

Phage cloning strategy two

• Mutagenesis PCR of pBlue with insert to remove KpnI restriction site
  • using turbo Pfu
  • elongation: 12,5min
  • DpnI digestion 2h at 37°C
  • transformation in TOP10, plated out on Cm plate

Phage cloning strategy one

• mutation of old insert in pBluescript
• pBluescript + insert was cut with BamHI --> the resulting backbone cut out of the gel and religated. With this vector 2 mutagenesis PCRs were done to eleminate the two remaining XbaI restriction sites.
• The resulting vector was digested with XbaI/XhoI to get the insert with the correct restriction sites for ligation into lambda phage

Characterization of oriT

• Inoculate the cells for conjugation test
  

5ml LB/chloramphenicol + glycerol stock Top10 pBAD 33
5ml LB/kanamycin/ampicilin +glycerol stock Top10 J01103+pUB307

Wednesday, 10/15/08

Phage cloning strategy two

• inoculation of KpnI mutagenesis PCR samples --> Miniprep
• Digestion with KpnI/AgeI
• Gel
• Gel purification kit

Characterization of oriT

• Quantitatively test for oriT
  

Donor: overnight culture Top10 oriT+pUB307 OD(600nm): 2.08
Recipient: overnight culture Top10 pBAD 33 OD(600nm): 2.24

• • Centrifuge 350ul overnight culture in 1.5ml eppi for 2min at 13000rpm, 8samples donor, 8samples recipient
  • Wash the pellet twice with LB medium
  • Resolve the pellet in 350ul LB medium
  • Centrifuge the washed recipient for 2min at 13000rpm, discard the fluid
  • Add the washed donor suspension
  • Vortex and resolve the pellet
  • Centrifuge the mix for 1min at 13000rpm
  • Resolve the pellet in 100ul LB
  • Put membrane filter on the LB agar
  • Pipett the suspension on membrane filter (8samples)
  • Incubate the plates with membrane filter at 37°C
  • Put directly one membrane filter into 1ml LB in an 1.5ml eppi
  • Vortex the eppi for 30sec, dilute for 10-5 and 10-6, plate them out on LB/Amp+Cm plates (0min)
  • After 6, 12, 18, 24, 30, 36, 42 min repeat the last two steps.
  • Negative control plates:
    • LB/Cm+Amp:

100ul donor overnight culture
100ul recipient overnight culture

• • Cell number determination
    • LB/Cm: 100ul 10-6 recipient overnight culture
    • LB/Kan+Amp: 100ul 10-6 donor overnight culture

• Result:
  • Negative control: negative
  • Colony on LB/Cm: 238 (Titer of recipient: 2.38e9/ml)
  • Colony on LB/Kan+Amp: 99 (Titer of donor: 0.99e9/ml)
  • Colony on other LB/Cm+Amp plates:
    • 10-5 dilute:
      

• • • 10-6 dilute:
      

• Inoculate the cells for conjugation test
  

5ml LB/chloramphenicol + glycerol stock Top10 pBAD 33
5ml LB/chloramphenicol + 1colony MG1655 pBAD 33
5ml LB/chloramphenicol + 1 colony stock DH5alpha pBAD 33
15ml LB/kanamycin/ampicilin +glycerol stock Top10 oriT+pUB307

Thursday, 10/16/08

Phage cloning strategy two

• overnight ligation of pBluescript/insert backbone, GFP and CmR

Characterization of oriT

• Quantitatively test for oriT
  

Donor: overnight culture Top10 oriT+pUB307 OD(600nm): 1.772
Recipient:
overnight culture Top10 pBAD 33 OD(600nm): 2.472
overnight culture MG1655 pBAD 33 OD(600nm): 4.412
overnight culture DH5alpha pBAD 33 OD(600nm): 3.876

• • Centrifuge overnight culture in 1.5ml eppi for 2min at 13000rpm, 24 samples donor, 400ul/sample; 8samples Top10 recipient, 340ul/sample; 8samples MG1655 recipient, 230ul/sample; 8samples DH5alpha recipient, 260ul/sample;
  • Wash the pellet twice with LB medium
  • Resolve the pellet in LB medium
  • Centrifuge the washed recipient for 2min at 13000rpm, discard the fluid
  • Add the washed donor suspension
  • Vortex and resolve the pellet
  • Centrifuge the mix for 1min at 13000rpm
  • Resolve the pellet in 100ul LB
  • Put membrane filter on the LB agar
  • Pipett the suspension on membrane filter (8samples x 3tests)
  • Incubate the plates with membrane filter at 37°C
  • Put directly one membrane filter into 1ml LB in an 1.5ml eppi
  • Vortex the eppi for 30sec, dilute for 10-5 and 10-6, plate them out on LB/Amp+Cm plates (0min)
  • After 6, 12, 18, 24, 30, 36, 42 min repeat the last two steps.
  • Negative control plates:
    • LB/Cm+Amp:
      

100ul donor overnight culture
100ul recipient overnight culture ( x 3)

• • Cell number determination
    
    • LB/Cm: 100ul 10-6 recipient overnight culture ( x 3)
    • LB/Kan+Amp: 100ul 10-6 donor overnight culture

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